疏水性阳离子聚合物PP80介导的抗肿瘤基因治疗
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摘要
安全、有效的基因载体是实现肿瘤基因治疗的基础。近年来，脂质体类、脂类、聚合物类等纳米基因载体，以其副作用小、安全、容易获得的优点逐步取代了病毒类基因载体。对高分子聚合物类载体进行修饰、改造、以提高其转染效率与生物相容性是目前材料合成研究中首要考虑的问题。超支化的25kDa的聚乙烯亚胺(PEI)是目前较为广泛使用的核酸传递载体，但是其生物不可降解性、高电荷密度导致高的细胞毒性，限制了其临床应用。通过对PEI进行接枝改性，以增强其生物相容性及转染效率是使其能够得到进一步应用的前提。一些经亲水性基团修饰的PEI极大地增强了自身的生物相容性，但却由于亲水性导致粒径增大，降低了其转染效率。因此，以疏水性修饰的材料逐渐被人们重视起来。材料经疏水性的聚酯类以及一些疏水性的氨基酸修饰后会有效地降低复合物表面的电位，增强与脂类细胞膜的亲和力，更有利于进入细胞内。已有研究表明，疏水性的阳离子聚合物能够有效的介导药物、siRNA与DNA的传递，并且具有较高的传递效率及较低的细胞毒性。我们前期工作中，分别通过NCA开环聚合和单体接枝的方法，向PEI25K引入不同数量的苯丙氨酸，制备出PEI-Phe聚合物（PP80），由于引入苯丙氨酸数量与分布合理，致使PEI-Phe有良好的DNA担载能力，并且粒径较小、血清稳定、转染效率较高。为了验证PP80作为基因载体在肿瘤基因治疗上的潜在应用性，本研究从评价PP80的生物安全性以及基因传递有效性两个方面开展如下工作。
首先，将带正电性的PP80与带负电性的报告基因加强型绿色荧光蛋白基因（pEGFPC1）通过静电吸引相复合，分别转染人宫颈癌、乳腺癌与肝癌细胞，结果显示，GFP在三种细胞内都有表达，说明PP80对三种比较难以转染的肿瘤细胞具有较好的亲和性，对HeLa细胞的转染效率约达到60%，而对MCF-7与HepG2的转染效率也分别为30%与20%。说明PP80是能够很好地靶向肿瘤细胞的一类基因载体。
为了验证PP80作为基因载体在生物体内的安全性，将PP80与质粒DNA复合，对其生物安全性进行评价。通过WST-1实验显示，PP80-pDNA复合物转染NIH3T3细胞7天内，经与对照组相比没有显示出明显的抑制细胞活力的现象。随后，分别将PP80与PP80-pDNA复合物加入到兔红细胞悬液中，结果显示，单独的PP80由于表面阳离子电荷的存在破坏了红细胞膜，引起了溶血现象，而PP80与质粒DNA复合后，中和了表面电荷未导致红细胞溶血。将该纳米基因复合物、对照质粒DNA和PBS分别注射到HeLa移植瘤内，作用22天后仍没有显示出影响肿瘤生长的现象。分离小鼠血清，分别检测了血清中的白介素-12与干扰素的表达水平，结果显示，该纳米基因复合物没有引起小鼠的炎症反应。分析血清中代表心、肝、肾等脏器的功能指标，如肌酸，谷丙转氨酶，谷草转氨酶，乳酸脱氢酶，肌苷，尿素氮的水平，结果显示，实验鼠体内酶的水平均与对照组无显著性差异，说明纳米基因复合物不影响各脏器的功能。家兔的热原质检测也说明了PP80-pDNA复合物不引起家兔的发热反应。此外，还通过微核试验检测了PP80-pDNA是否能引起小鼠的急性遗传毒性，结果显示该复合物没有引起小鼠染色体畸变，初步判定不具有遗传毒性。以上结果表明，基因载体PP80具有较好的生物相容性并且能够很好地介导外源基因在肿瘤细胞内表达。
为了进一步将PP80应用到肿瘤基因治疗水平，我们首先建立了HeLa裸鼠移植瘤模型，通过凝胶阻滞实验及粒径与电势分析，摸索了适用于肿瘤组织转染的最佳的纳米基因结合比例。结果显示，当PP80与质粒DNA质量比为0.5:1时，能够介导绿色荧光蛋白基因及荧光素酶报告基因在肿瘤内大量表达。随后，将PP80与持续诱导细胞凋亡的抗肿瘤基因rev-casp-3通过静电吸引相复合，通过实体瘤注射的方式，注射到肿瘤内，每2天治疗一次，共治疗4次，结果显示PP80介导的rev-casp-3能够有效的抑制HeLa移植瘤的生长。进一步分析了肿瘤组织内的Caspase3下游的靶基因核糖核酸聚合酶1（PARP1），发现该酶被切割成85kDa与25kDa两种形式，失去了修复受损DNA的能力，致使肿瘤走向凋亡。TUNEL实验进一步验证了PP80介导的rev-casp-3诱导了肿瘤细胞凋亡。同时，也检测了各肿瘤组织中细胞增殖的情况，发现过表达rev-casp-3组细胞增殖的Marker Ki-67阳性率明显降低，推测rev-casp-3是通过诱导肿瘤凋亡来抑制肿瘤细胞生长及肿瘤增殖。此外，该凋亡现象也在对体外培养的HeLa细胞中过表达rev-casp-3的实验中得到了进一步的验证。
为了进一步验证PP80作为基因载体的广谱性与有效性，我们又将PP80与抗酸性成纤维细胞生长因子(FGF-1)的单链抗体基因scFv1C9相结合，检测PP80介导scFv1C9基因在肿瘤内表达并发挥作用。scFv1C9为本实验室通过基因工程的手段获得的特异性结合FGF-1的单链抗体基因，通过上调细胞周期负调控因子p21的表达，从而影响周期蛋白激酶CDK2的表达，引起细胞周期G0/G1期阻滞，抑制细胞增殖。将PP80与scFv1C9通过静电吸引相复合注射到裸鼠HepG2移植瘤内，结果显示，PP80介导的scFv1C9能够抑制肝癌移植瘤的生长。说明PP80是一个具有广谱性、有效性的基因传递载体。
综上所述，本研究中通过对疏水性阳离子聚合物作为基因载体的安全性与有效性两个方面做出研究。通过生物安全性评价方法初步证明PP80具有较好的生物相容性，适用于体外及体内水平应用。此外，PP80能够介导抗肿瘤基因rev-casp-3与scFv1C9在肿瘤内表达并抑制小鼠移植瘤的生长。以上研究结果表明，阳离子聚合物PP80有望成为癌症基因治疗的传递载体。
Safety and efficacy of gene carriers were regarded as the basis for tumor gene therapy.Recently, liposomes, lipoid and polymer had been used as gene carries to substitute virusvector because of showing advantages such as lower adverse effect and easier obtaining.However, modification and alteration for the polymers to improve its transfection efficiencyand biocompatibility were primarily considered in material synthesis area. Branchedpolyethylenimine, especially PEI25kDa, has generally been accepted as gene carrier,howerver, its nonbiodegradability, high cytotoxicity from much charge density limited itsapplication in clinical trials. Through Modification of PEI to improve the biocompatibility andtransfection efficiency was regarded the foundation to further apply it. Some polymer graftedby hydrophilic segments showed favorable biocompatibility but its transfection efficiency wasreduced due to the increasing of particle size. Therefore, a series of hydrophobic modifiedpolymers were gradually pay noticed. The hydrophobic modified materials possessed betteraffinity to the lipid surface of cell membrane which promoted its entry into the cells. Studieshave shown that hydrophobic cationic polymers could effetively mediate the delivery of drug,siRNA and plasmid DNA with low cell toxicity. In our previous study, we synthesized PPhenby NCA ring opening polymerization and monomer grafting of phenylalanines (Phe) toPEI25K which possessed small particle size and good stability. In order to expore thepotential of PP80as a gene carrier for tumor treatment in vivo, it is necessary to assess thesafety and efficacy of gene carriers.
Firstly, PP80was complexed with GFP gene and separately added into different kinds ofadherent tumor cells grown on six-well plate. As the results showed GFP was expressed inHeLa, MCF-7and HepG2cells. The transfected efficiency was up to60%in HeLa cells but30%and20%separately in MCF-7and HepG2celles. It indicated that PP80possessed goodaffinity for tumor cell, especially for HeLa cells. These results demonstrated that PP80was aneffective gene carrier for targeting tumor cells.
In order to verify the safety of PP80as gene carrier, the biocompatibility wasinvestigated in vitro and in vivo. As shown in WST-1experiments, there were no obviousinfluences on NIH3T3cell growth after7days incubation with PP80-pDNA complex.Subsequently, the complex was added into fresh rabbit blood incubating for30min, theresults showed that only PP80strongly induced hemolysis but not PP80-pDNA complex. Inaddition, this complex was also shown no inhibition on the tumor growth when injected intoHeLa xenograft compared with nude pDNA or PBS. To investigate the possible inflammatory reaction induced by PP80, the levels of cytokines were measured. The results showed thatthere were no significant increases in either mIL-12p70or IFN-g in any groups. Meanwhile,CK, LDH and CK-MB, ALT and AST, as well as Cr and BUN, were quantitatively examinedto evaluate the functions of heart, liver and kidney, respectively. As shown in Table1, none ofthese enzymes were significantly changes in all mice. Moreover, pyrogenetic reaction fromrabbit treating with complex was also not observed. Finally, the complex was injected intomice peritoneal cavity to examine its genetoxic by micronucleus tests, from the resultsverdicted that there was no inhibition chromosomal aberration. Taken together, the aboveresults demonstrated that PP80was safe for the delivery of pDNA in vitro and in vivo.
The above study exhibited that PP80was well biocompatible with animals and effectivelymediated gene expression in tumor. To verify the further application in gene therapy, thecomplexed ratio of PP80and pDNA was optimized by assessing the gene expression in HeLaxenograft. From the gel retardation and size assay, the luciferase was abundantly expressed inthe tumor tissue which was injected PP80/pDNA complex at the ratio of0.5. Subsequently,tumor growth rate was decreased significantly by injecting PP80-pKH3-rev-casp-3complex.In the process of therapy, poly (ADP-ribose) polymerase (PARP1) was cleaved into twofragments of85kDa and25kDa, which lost the repaired function for the damaged DNA due tothe overexpression of rev-casp-3, and at last induced cell or tumor apoptosis. Meanwhile, thestatus of cell proliferation in tumor tissue was also detected, discovering that the Ki-67positive cells were strongly decreasded in rev-casp-3overexpressing group, it is supposed thatthe slow growth of tumor was possibly due to cell apoptosis inducing by rev-casp-3. TUNELanalysis also provided evidence that PP880deliveried rev-casp-3can effectively inhibit thetumoral growth mainly though apoptotic pawthway. This apoptosic phenomenon was furtherconfirmed by overexpressing rev-casp-3in HeLa cells.
To further verify the extensive applicability of PP80as a gene vector, we also combinedPP80with a single-chain antibody gene (scFv1C9) against fibroblast growth factor1(FGF-1)to cure tumor. In our study, it was demonstrated that scFv1C9resulted in the G0/G1areest byregulation of the expression of p21and CDK2. Futhermore, PP880deliveried scFv1C9caneffectively inhibit HepG2xenograft growth.
In summary, these work mainly foucus on developing the safety and effectiveness ofhydrophobic cationic as gene carrier. First of all, through biological safety evaluation, wedemonstrated PP80was safety and can be applied in vitro and in vivo. In addition, PP80caneffectively deliver rev-casp-3and scFv1C9expression in tumor and inhibit xenogaraft growth.These works have provided that PP80was a potential gene carrier for cancer gene therapy.
首先，将带正电性的PP80与带负电性的报告基因加强型绿色荧光蛋白基因（pEGFPC1）通过静电吸引相复合，分别转染人宫颈癌、乳腺癌与肝癌细胞，结果显示，GFP在三种细胞内都有表达，说明PP80对三种比较难以转染的肿瘤细胞具有较好的亲和性，对HeLa细胞的转染效率约达到60%，而对MCF-7与HepG2的转染效率也分别为30%与20%。说明PP80是能够很好地靶向肿瘤细胞的一类基因载体。
为了验证PP80作为基因载体在生物体内的安全性，将PP80与质粒DNA复合，对其生物安全性进行评价。通过WST-1实验显示，PP80-pDNA复合物转染NIH3T3细胞7天内，经与对照组相比没有显示出明显的抑制细胞活力的现象。随后，分别将PP80与PP80-pDNA复合物加入到兔红细胞悬液中，结果显示，单独的PP80由于表面阳离子电荷的存在破坏了红细胞膜，引起了溶血现象，而PP80与质粒DNA复合后，中和了表面电荷未导致红细胞溶血。将该纳米基因复合物、对照质粒DNA和PBS分别注射到HeLa移植瘤内，作用22天后仍没有显示出影响肿瘤生长的现象。分离小鼠血清，分别检测了血清中的白介素-12与干扰素的表达水平，结果显示，该纳米基因复合物没有引起小鼠的炎症反应。分析血清中代表心、肝、肾等脏器的功能指标，如肌酸，谷丙转氨酶，谷草转氨酶，乳酸脱氢酶，肌苷，尿素氮的水平，结果显示，实验鼠体内酶的水平均与对照组无显著性差异，说明纳米基因复合物不影响各脏器的功能。家兔的热原质检测也说明了PP80-pDNA复合物不引起家兔的发热反应。此外，还通过微核试验检测了PP80-pDNA是否能引起小鼠的急性遗传毒性，结果显示该复合物没有引起小鼠染色体畸变，初步判定不具有遗传毒性。以上结果表明，基因载体PP80具有较好的生物相容性并且能够很好地介导外源基因在肿瘤细胞内表达。
为了进一步将PP80应用到肿瘤基因治疗水平，我们首先建立了HeLa裸鼠移植瘤模型，通过凝胶阻滞实验及粒径与电势分析，摸索了适用于肿瘤组织转染的最佳的纳米基因结合比例。结果显示，当PP80与质粒DNA质量比为0.5:1时，能够介导绿色荧光蛋白基因及荧光素酶报告基因在肿瘤内大量表达。随后，将PP80与持续诱导细胞凋亡的抗肿瘤基因rev-casp-3通过静电吸引相复合，通过实体瘤注射的方式，注射到肿瘤内，每2天治疗一次，共治疗4次，结果显示PP80介导的rev-casp-3能够有效的抑制HeLa移植瘤的生长。进一步分析了肿瘤组织内的Caspase3下游的靶基因核糖核酸聚合酶1（PARP1），发现该酶被切割成85kDa与25kDa两种形式，失去了修复受损DNA的能力，致使肿瘤走向凋亡。TUNEL实验进一步验证了PP80介导的rev-casp-3诱导了肿瘤细胞凋亡。同时，也检测了各肿瘤组织中细胞增殖的情况，发现过表达rev-casp-3组细胞增殖的Marker Ki-67阳性率明显降低，推测rev-casp-3是通过诱导肿瘤凋亡来抑制肿瘤细胞生长及肿瘤增殖。此外，该凋亡现象也在对体外培养的HeLa细胞中过表达rev-casp-3的实验中得到了进一步的验证。
为了进一步验证PP80作为基因载体的广谱性与有效性，我们又将PP80与抗酸性成纤维细胞生长因子(FGF-1)的单链抗体基因scFv1C9相结合，检测PP80介导scFv1C9基因在肿瘤内表达并发挥作用。scFv1C9为本实验室通过基因工程的手段获得的特异性结合FGF-1的单链抗体基因，通过上调细胞周期负调控因子p21的表达，从而影响周期蛋白激酶CDK2的表达，引起细胞周期G0/G1期阻滞，抑制细胞增殖。将PP80与scFv1C9通过静电吸引相复合注射到裸鼠HepG2移植瘤内，结果显示，PP80介导的scFv1C9能够抑制肝癌移植瘤的生长。说明PP80是一个具有广谱性、有效性的基因传递载体。
综上所述，本研究中通过对疏水性阳离子聚合物作为基因载体的安全性与有效性两个方面做出研究。通过生物安全性评价方法初步证明PP80具有较好的生物相容性，适用于体外及体内水平应用。此外，PP80能够介导抗肿瘤基因rev-casp-3与scFv1C9在肿瘤内表达并抑制小鼠移植瘤的生长。以上研究结果表明，阳离子聚合物PP80有望成为癌症基因治疗的传递载体。
Safety and efficacy of gene carriers were regarded as the basis for tumor gene therapy.Recently, liposomes, lipoid and polymer had been used as gene carries to substitute virusvector because of showing advantages such as lower adverse effect and easier obtaining.However, modification and alteration for the polymers to improve its transfection efficiencyand biocompatibility were primarily considered in material synthesis area. Branchedpolyethylenimine, especially PEI25kDa, has generally been accepted as gene carrier,howerver, its nonbiodegradability, high cytotoxicity from much charge density limited itsapplication in clinical trials. Through Modification of PEI to improve the biocompatibility andtransfection efficiency was regarded the foundation to further apply it. Some polymer graftedby hydrophilic segments showed favorable biocompatibility but its transfection efficiency wasreduced due to the increasing of particle size. Therefore, a series of hydrophobic modifiedpolymers were gradually pay noticed. The hydrophobic modified materials possessed betteraffinity to the lipid surface of cell membrane which promoted its entry into the cells. Studieshave shown that hydrophobic cationic polymers could effetively mediate the delivery of drug,siRNA and plasmid DNA with low cell toxicity. In our previous study, we synthesized PPhenby NCA ring opening polymerization and monomer grafting of phenylalanines (Phe) toPEI25K which possessed small particle size and good stability. In order to expore thepotential of PP80as a gene carrier for tumor treatment in vivo, it is necessary to assess thesafety and efficacy of gene carriers.
Firstly, PP80was complexed with GFP gene and separately added into different kinds ofadherent tumor cells grown on six-well plate. As the results showed GFP was expressed inHeLa, MCF-7and HepG2cells. The transfected efficiency was up to60%in HeLa cells but30%and20%separately in MCF-7and HepG2celles. It indicated that PP80possessed goodaffinity for tumor cell, especially for HeLa cells. These results demonstrated that PP80was aneffective gene carrier for targeting tumor cells.
In order to verify the safety of PP80as gene carrier, the biocompatibility wasinvestigated in vitro and in vivo. As shown in WST-1experiments, there were no obviousinfluences on NIH3T3cell growth after7days incubation with PP80-pDNA complex.Subsequently, the complex was added into fresh rabbit blood incubating for30min, theresults showed that only PP80strongly induced hemolysis but not PP80-pDNA complex. Inaddition, this complex was also shown no inhibition on the tumor growth when injected intoHeLa xenograft compared with nude pDNA or PBS. To investigate the possible inflammatory reaction induced by PP80, the levels of cytokines were measured. The results showed thatthere were no significant increases in either mIL-12p70or IFN-g in any groups. Meanwhile,CK, LDH and CK-MB, ALT and AST, as well as Cr and BUN, were quantitatively examinedto evaluate the functions of heart, liver and kidney, respectively. As shown in Table1, none ofthese enzymes were significantly changes in all mice. Moreover, pyrogenetic reaction fromrabbit treating with complex was also not observed. Finally, the complex was injected intomice peritoneal cavity to examine its genetoxic by micronucleus tests, from the resultsverdicted that there was no inhibition chromosomal aberration. Taken together, the aboveresults demonstrated that PP80was safe for the delivery of pDNA in vitro and in vivo.
The above study exhibited that PP80was well biocompatible with animals and effectivelymediated gene expression in tumor. To verify the further application in gene therapy, thecomplexed ratio of PP80and pDNA was optimized by assessing the gene expression in HeLaxenograft. From the gel retardation and size assay, the luciferase was abundantly expressed inthe tumor tissue which was injected PP80/pDNA complex at the ratio of0.5. Subsequently,tumor growth rate was decreased significantly by injecting PP80-pKH3-rev-casp-3complex.In the process of therapy, poly (ADP-ribose) polymerase (PARP1) was cleaved into twofragments of85kDa and25kDa, which lost the repaired function for the damaged DNA due tothe overexpression of rev-casp-3, and at last induced cell or tumor apoptosis. Meanwhile, thestatus of cell proliferation in tumor tissue was also detected, discovering that the Ki-67positive cells were strongly decreasded in rev-casp-3overexpressing group, it is supposed thatthe slow growth of tumor was possibly due to cell apoptosis inducing by rev-casp-3. TUNELanalysis also provided evidence that PP880deliveried rev-casp-3can effectively inhibit thetumoral growth mainly though apoptotic pawthway. This apoptosic phenomenon was furtherconfirmed by overexpressing rev-casp-3in HeLa cells.
To further verify the extensive applicability of PP80as a gene vector, we also combinedPP80with a single-chain antibody gene (scFv1C9) against fibroblast growth factor1(FGF-1)to cure tumor. In our study, it was demonstrated that scFv1C9resulted in the G0/G1areest byregulation of the expression of p21and CDK2. Futhermore, PP880deliveried scFv1C9caneffectively inhibit HepG2xenograft growth.
In summary, these work mainly foucus on developing the safety and effectiveness ofhydrophobic cationic as gene carrier. First of all, through biological safety evaluation, wedemonstrated PP80was safety and can be applied in vitro and in vivo. In addition, PP80caneffectively deliver rev-casp-3and scFv1C9expression in tumor and inhibit xenogaraft growth.These works have provided that PP80was a potential gene carrier for cancer gene therapy.
引文
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